CN113388034A

CN113388034A - Antigenic peptide of lipoprotein (a), antibody thereof and use thereof

Info

Publication number: CN113388034A
Application number: CN202010167325.4A
Authority: CN
Inventors: 林春娇; 黄斌; 张裕平
Original assignee: Shenzhen Mindray Bio Medical Electronics Co Ltd
Current assignee: Shenzhen Mindray Bio Medical Electronics Co Ltd
Priority date: 2020-03-11
Filing date: 2020-03-11
Publication date: 2021-09-14

Abstract

The present invention relates to lipoprotein (a) antibodies or antigen-binding fragments thereof that specifically bind to one or more KIV antigen peptides selected from the group consisting of the peptides represented by SEQ ID NOS: 1-3. The use of such an antibody makes it possible to accurately detect the amount of lipoprotein (a) in a sample without the need to establish a curve using a standard. Furthermore, the invention relates to corresponding antigenic peptides, nucleic acid molecules, fusion proteins and methods.

Description

Antigenic peptide of lipoprotein (a), antibody thereof and use thereof

Technical Field

The invention relates to the field of immunodetection, in particular to an antigenic peptide of lipoprotein (a) and an antibody specifically bound with the antigenic peptide.

Background

Lipoprotein (a) is mainly synthesized in the liver, and the detection of lipoprotein (a) can be combined with the symptoms of a patient and other related diagnostic results (such as triglyceride, cholesterol, high density lipoprotein, low density lipoprotein, homotypic cysteine, and the like) to diagnose cardiovascular diseases. That is, accurate detection of lipoprotein (a) would be beneficial in the adjuvant determination of cardiovascular disease. Like other lipoproteins, lipoprotein (a) includes a fat core containing cholesterol, triglycerides, phospholipids, and the like. The fat core is coated with 2 protein chains consisting of apoB and apo (a) which characterizes lipoprotein (a), and linked by disulfide bonds. Therefore, the detection of lipoprotein (a) can be performed only for apo (a) thereof.

apo (a) is a protein formed by the linkage of many small "loops" (also known as loop-cake structures) whose cake-loop structures can be classified into KIV-1 to KIV-10 types according to amino acid sequence differences. Other forms, except KIV-2, have only one ring cake structure, while KIV-2 repeats at different numbers of rings in each apo (a), which may range from 3 to 40 copies, resulting in apo (a) molecular weights varying from 187000 to 662000 for different individuals. Therefore, the heterogeneity of KIV-2 relative to molecular mass decreases the accuracy of the lipoprotein (a) immunoassay. Specifically, if the antibody recognizes multiple copies of the KIV-2 cake-loop region, the detection result of the lipoprotein (a) obtained is low if the apo (a) particle size of the test object is smaller than that of the calibrator; otherwise, the result will be higher.

To overcome the difficulty in accurately reflecting the level of lipoprotein (a), 5 calibrators of different particle sizes were used in both roche and electrochemical studies. On one hand, the accuracy of the method strongly depends on the curve established by the calibration product, and on the other hand, the process for preparing the calibration product is very complicated and the cost is high.

To avoid the introduction of calibrators of multiple gradients, particle concentration of lipoprotein (a) could theoretically be detected using antibodies that recognize regions with a single copy number loop-cake on each apo (a) (i.e., non-KIV-2 regions).

In contrast, some researchers have tried to obtain polyclonal antibodies by immunizing with recombinant KIV-1 cake loops as antigen, but because the degree of similarity between the antigen used and the KIV-2 cake is high, the obtained polyclonal antibodies still have affinity for the KIV-2 cake loops, and therefore, antibodies that are not KIV-2 specific in the true sense cannot be obtained, and the level of lipoprotein (a) cannot be accurately reflected.

In addition, some researchers have tried to screen non-KIV-2 specific antibodies using native lipoprotein (a) as an immunizing antigen, but because of the multiple copy number of KIV-2 and its high similarity to other KIV loop cakes, the efficiency of screening non-KIV-2 specific antibodies using native lipoprotein (a) as an immunizing antigen is low, and thus antibodies that accurately reflect the level of lipoprotein (a) cannot be obtained efficiently.

Accordingly, in the field of lipoprotein (a) detection, there is a strong demand for rapid and convenient acquisition of non-KIV-2 specific antibodies.

Disclosure of Invention

To facilitate screening of non-KIV-2 specific antibodies, the present inventors have studied the apo (a) structure of lipoprotein (a). In a first aspect, the present invention provides an antigenic peptide having an amino acid sequence as shown in one or more of SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3.

Using the antigenic peptides of the invention and following conventional methods of antibody production, antibodies that specifically bind to the KIV-6/7, KIV-8 or KIV-9 region, but not the KIV-2 region, can be obtained by immunizing only a few animals. In addition, such antibodies can be used for the detection of lipoprotein (a), and have high comparability as compared with commercially available products. Further, the antibody obtained using the antigenic peptide of the present invention satisfies the performance requirements of the reagent with respect to the degree of deviation.

In a specific embodiment, the amino acid sequence of the antigenic peptide of the invention is set forth in SEQ ID NO 1.

In a specific embodiment, the amino acid sequence of the antigenic peptide of the invention is set forth in SEQ ID NO 2.

In a preferred embodiment, the amino acid sequence of the antigenic peptide of the invention is as shown in SEQ ID NO 3.

In a specific embodiment, the antigenic peptide of the present invention may also be a combination of any two or more of the antigenic peptides having the amino acid sequences described above as immunogens. For example: the antigenic peptide having the amino acid SEQ ID NO. 1 and the antigenic peptide having the amino acid SEQ ID NO. 3 are used together as an immunogen.

It should be noted that the present invention further improves the efficiency of screening for non-KIV-2 specific anti-lipoprotein (a) antibodies by selecting the antigenic peptide shown in SEQ ID NO. 3, and as demonstrated in the examples section below, 6 animals were immunized by preparing the antigen using the antigenic peptide shown in SEQ ID NO. 3, and the resulting 6 immune sera were all positive for KIV-9 and negative for KIV-2. Further, in the case of detecting lipoprotein (a) using an antibody screened with the antigen peptide represented by SEQ ID NO. 3, it has very high comparability to commercially available products.

In a second aspect, the present invention provides a fusion protein comprising an antigenic peptide of the invention.

In some embodiments, the fusion protein of the invention further comprises a polypeptide selected from an affinity tag.

In some embodiments, the fusion protein of the invention further comprises a protein carrier.

In some embodiments, the fusion protein of the invention further comprises a polypeptide that targets the fusion protein to a predetermined location.

In a third aspect, the invention provides a nucleic acid molecule encoding an antigenic peptide of the invention or a fusion protein of the invention.

In a fourth aspect, the present invention provides an expression vector comprising the nucleic acid molecule of the third aspect of the invention.

In a fifth aspect, the present invention provides a host cell comprising a nucleic acid molecule according to the third aspect of the invention or an expression vector according to the fourth aspect of the invention.

In a sixth aspect, the present invention provides a method for obtaining an anti-lipoprotein (a) antibody that is not specific for KIV-2, using the antigenic peptide of the present invention as an antigen.

In one variant of the method for obtaining an anti-lipoprotein (a) antibody which is not specific for KIV-2, the method utilizes as an antigen a fusion protein of an antigenic peptide of the invention and a protein carrier.

In a seventh aspect, the present invention provides a method for producing an antibody, which uses the antigenic peptide of the present invention as an immunogen to produce an antibody.

In one variation of the method for producing an antibody, the method utilizes a fusion protein of the antigenic peptide of the present invention and a protein carrier as an immunogen to produce the antibody.

In an eighth aspect, the invention provides an antibody or antigen-binding fragment thereof that specifically binds to one or more KIV antigen peptides selected from the group consisting of the KIV antigen peptides represented by SEQ ID NOs 1-3.

Alternatively, the antibody or antigen-binding fragment thereof of the invention is produced using the antigenic peptide of the first aspect of the invention.

It is understood that the antigenic peptides shown by SEQ ID NO. 1 are located on the KIV-6 region and the KIV-7 region, and that when screening is performed using the antigenic peptides shown by SEQ ID NO. 1, antibodies that specifically bind to the corresponding epitopes on KIV-6 and KIV-7 can be obtained. The antigenic peptide shown in SEQ ID NO. 2 is located on the KIV-8 region, and when the antigenic peptide shown in SEQ ID NO. 2 is used for screening, an antibody specifically bound with the corresponding epitope on KIV-8 can be obtained. The antigenic peptide shown by SEQ ID NO. 3 is located on the KIV-9 region, and when the antigenic peptide shown by SEQ ID NO. 3 is used for screening, an antibody which is specifically combined with the corresponding epitope on KIV-9 can be obtained.

In some embodiments, the antibodies or antigen-binding fragments of the invention do not specifically bind to the KIV-2 antigen peptide as set forth in SEQ ID NO. 4.

In a preferred embodiment, the antibodies or binding fragments thereof of the invention specifically bind to the KIV antigen peptide as set forth in SEQ ID NO 3.

In some embodiments, the antibody of the invention may be selected from the group consisting of monoclonal antibodies, polyclonal antibodies, bispecific antibodies, chimeric antibodies, humanized antibodies, and single chain antibodies.

In a specific embodiment, the antibody of the invention is a monoclonal antibody.

In a specific embodiment, the antibody of the invention is a polyclonal antibody.

In particular embodiments, the antibody may be murine, rabbit, or ovine.

In a ninth aspect, the invention provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the invention.

In a tenth aspect, the present invention provides a kit for detecting lipoprotein (a) comprising an antibody or antigen-binding fragment thereof of the present invention.

In some embodiments, the kit comprises an antibody or antigen-binding fragment thereof of the invention, and wherein the antibody or antigen-binding fragment thereof is coated on a solid support.

In other embodiments, a kit comprises an antibody or antigen-binding fragment thereof of the invention, and wherein the antibody or antigen-binding fragment thereof carries a detectable label.

In still other embodiments, a kit comprises a first antibody or antigen-binding fragment thereof of the invention and a second antibody or antigen-binding fragment thereof of the invention, and wherein the first antibody or antigen-binding fragment thereof binds a KIV antigen peptide that is different from the KIV antigen peptide of the second antibody or antigen-binding fragment thereof.

In still other embodiments, a kit comprises an antibody or antigen-binding fragment thereof of the invention, and wherein the antibody is not coated on a solid support and does not carry a detectable label.

In the present invention, the expressions "first" and "second", etc. are used for descriptive purposes only to distinguish between the defined substances, and not to define an order or primary or secondary in any way.

In an eleventh aspect, the present invention provides a method for detecting lipoprotein (a), comprising the steps of:

a) contacting a sample derived from a subject with an antibody or antigen-binding fragment thereof of the invention; and

b) determining whether lipoprotein (a) is present in the sample or determining the level of lipoprotein (a) present in the sample.

In some embodiments, the antibody or antigen-binding fragment thereof is coated on a solid support.

In other embodiments, the antibody or antigen-binding fragment thereof carries a detectable label.

In still other embodiments, the antibody or antigen-binding fragment thereof is not coated on a solid support and does not carry a detectable label.

In an exemplary embodiment, the antibody or antigen binding fragment thereof is used in a two-antibody sandwich assay.

In a twelfth aspect, the present invention provides the use of an antibody or antigen-binding fragment thereof in the preparation of a kit for the detection of lipoprotein (a).

In a thirteenth aspect, the present invention provides a composition comprising one or more KIV antigen peptides selected from the group consisting of the KIV antigen peptides represented by SEQ ID NOs 1-3, and the KIV-2 antigen peptide represented by SEQ ID NO 4.

In a fourteenth aspect, there is provided the use of an antibody or antigen-binding fragment thereof of the eighth aspect of the invention in the manufacture of a kit for use in a method for assessing cardiovascular disease, the method comprising the steps of:

a) contacting a sample derived from a subject with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6; and

Through the research on the apo (a) chain in the lipoprotein (a), the invention discovers and confirms the antigenic peptide sequences shown in SEQ ID NO 1 to 3 in the apo (a) chain for the first time, and through the use of the antigenic peptide sequences, antibodies which specifically bind to KIV-6/7, KIV-8 or KIV-9 regions with only a single copy can be produced more conveniently and rapidly, and the antibodies do not specifically bind to the KIV-2 region. When such an antibody is used for detecting lipoprotein (a), accuracy of the detection result can be improved, and the present invention greatly improves the efficiency of screening such an antibody. In addition, the scheme of the invention avoids the introduction of multi-concentration standard substances in the detection process of the lipoprotein (a), and reduces the cost of detection reagents.

Drawings

FIG. 1 shows the results of the lipoprotein (a) test kit prepared with antibody # A1 aligned with commercial reagents;

FIG. 2 shows the relationship between particle size and absolute deviation in the case of microspheres coated with antibody # A1;

FIG. 3 shows absolute deviations corresponding to 0 to 300nmol/L of a reference substance in the case of preparing a lipoprotein (a) detection kit using the antibody # A1, wherein the evaluation of the concentration ≦ 100nmol/L is based on the absolute deviation;

FIG. 4 shows relative deviations corresponding to 0 to 300nmol/L of a reference substance, and relative deviations corresponding to an evaluation of a reference substance at a concentration of more than 100nmol/L, in the case of preparing a lipoprotein (a) detection kit using the antibody # A1;

FIG. 5 shows the results of the lipoprotein (a) test kit prepared with antibody # B4 aligned with commercial reagents;

FIG. 6 shows the relationship between particle size and absolute deviation in the case of microspheres coated with antibody # B4;

FIG. 7 shows absolute deviations corresponding to 0 to 300nmol/L of a reference substance in the case of preparing a lipoprotein (a) detection kit using the antibody # B4, wherein the evaluation of the concentration ≦ 100nmol/L is based on the absolute deviation;

FIG. 8 shows relative deviations corresponding to 0 to 300nmol/L of a reference substance, and relative deviations corresponding to reference substances having a concentration of more than 100nmol/L are evaluated in the case of preparing a lipoprotein (a) detection kit using the antibody # B4;

FIG. 9 shows the results of the lipoprotein (a) test kit prepared with antibody # C3 aligned with commercial reagents;

FIG. 10 shows the relationship between particle size and absolute deviation in the case of microspheres coated with antibody # C3;

FIG. 11 shows absolute deviations corresponding to 0 to 300nmol/L of a reference substance in the case of preparing a lipoprotein (a) detection kit using the antibody # C3, wherein the evaluation of concentration ≦ 100nmol/L is based on the absolute deviation;

FIG. 12 shows relative deviations corresponding to 0 to 300nmol/L of the reference substance and relative deviations corresponding to the evaluation of the reference substance at a concentration of more than 100nmol/L in the case of preparing the lipoprotein (a) detection kit using the antibody # C3.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.

As used herein, the term "antibody" refers to immunoglobulin molecules, including but not limited to monoclonal antibodies, polyclonal antibodies, bispecific antibodies, chimeric antibodies, humanized antibodies, and single chain antibodies; furthermore, it relates to antibodies produced/synthesized recombinantly or synthetically. Antibody, if at greater than or equal to about 10⁴M^-1Preferably greater than or equal to about 10⁵M^-1More preferably greater than or equal to about 10⁶M^-1And more preferably greater than or equal to about 10⁷M^-1Is defined as being "immunospecific" or specifically binding when it binds to an antigenic peptide.

As used herein, the term "monoclonal antibody" refers to an antibody that is a highly homogeneous antibody raised against only a particular epitope of a B cell clone, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific for a single antigenic site. Monoclonal antibodies are advantageous because they can be obtained by culture of hybridoma cell lines and are substantially free of contamination by other immunoglobulins.

As used herein, the term "polyclonal antibody" refers to the blood supernatant collected after the measurement of titer by injecting an antigen directly into an animal without preparing hybridoma cells for immunization. Optionally, after obtaining the supernatant, further purification is performed to obtain a polyclonal antibody.

As used herein, the term "antigen-binding fragment" generally includes the antigen-binding region, at least a portion of the light chain and/or heavy chain variable region (e.g., the six CDRs), of the parent antibody that retains at least some of the binding specificity of the parent antibody. Examples of antigen binding fragments include, but are not limited to, Fab ', F (ab')2, and Fv fragments.

Preferably, an antigen-binding fragment refers to the antigen-binding region, the light and heavy chain variable regions, or the six CDRs of an antibody.

The antibodies of the invention also include derivatives of antibodies, and as used herein, "derivatives of antibodies" refer to antibodies that may include conservative amino acid substitutions (also referred to as "conservative variants") that have substantially no altered biological activity as compared to the parent antibody.

Polyclonal antibodies can be prepared by any of a variety of techniques known to those of ordinary skill in the art. In one such technique, an immunogen comprising an antigenic peptide of the present invention (e.g., an antigenic peptide conjugated to a protein carrier) is first injected into an appropriate animal, preferably with one or more booster immunizations performed according to a predetermined schedule, and then periodically bled from the animal. Polyclonal antibodies specific for the antigenic peptides of the invention can then be purified from these sera by purification techniques, such as Protein A affinity purification.

Likewise, monoclonal antibodies can be prepared by any of a variety of techniques known to those of ordinary skill in the art. For example, these methods may include: an immortalized cell line capable of producing antibodies with the desired specificity is prepared. Such cell lines can be generated, for example, from spleen cells obtained from immunized animals. The spleen cells are then immortalized, for example, by fusing myeloma cell fusion partners (e.g., partners homologous to the animal being immunized). For example, spleen cells and myeloma cells can be combined for several minutes using a membrane fusion promoter (e.g., polyethylene glycol or a non-ionic detergent) and then plated at low density on selective media that support hybrid cell growth but not myeloma cell growth. After a sufficient period of time, typically about 1-2 weeks, heterozygote colonies are observed. Individual colonies were selected and tested for binding activity to the antigen peptide. Hybridomas having high reactivity and specificity are preferred. Monoclonal antibodies can be isolated from the supernatant of growing hybridoma colonies. In addition, various techniques can be used to increase the yield, such as injecting the hybridoma cell line into the abdominal cavity of a suitable vertebrate host. The monoclonal antibodies are then harvested from the ascites fluid or blood. Contaminants can be removed from the antibody by conventional techniques, such as chromatography, gel filtration, precipitation or extraction. For example, antibodies can be purified by chromatography on immobilized Protein G or Protein A using standard techniques.

The detection kit of the present invention may take various forms, for example, a strip, a cartridge containing various reagents required for the test, a microfluidic chip, etc., and the kit may be manufactured according to standard procedures known to those skilled in the art.

Kits of the invention may include containers, chips, instructions for use, buffers, immunological aids, and/or other materials, structures, and/or reagents as desired for performing the diagnosis/assay.

In the examples, the kit of the present invention is described by taking an example of increasing transmittance with latex, but the kit of the present invention is not limited thereto.

The kits of the invention include non-KIV-2 specific antibodies generated from animals immunized with the antigenic peptide of the invention and may be presented in a manner conventional in the art, e.g., in a dissolved or dried form in a container, coated on a solid support (e.g., a membrane, a plate, beads (e.g., magnetic beads), microspheres (such as carboxyl microspheres, amino microspheres, chloromethyl microspheres, physisorption microspheres, etc.), in a dissolved or dried form in a chamber of a chip, although the invention is not limited thereto.

The antibody of the present invention can be used at a concentration of about 10 to 100. mu.g/ml.

Other reagents required for diagnosis/detection in the kit of the present invention include, but are not limited to, anti-lipoprotein (a) antibodies other than the antibody of the present invention, anti-human antibodies, and the like. The other reactants mentioned above may be present in a manner conventional in the art, for example, in dissolved or dried form in a container, coated on a solid support, in dissolved or dried form in a chamber of a chip, but the present invention is not limited thereto.

Other materials required for performing an assay in a kit of the invention include, but are not limited to, materials for sampling, materials for performing controls, and/or materials for observing the course or result of the assay.

Other reagents required for performing the assay in the kits of the invention include, but are not limited to, detergents, visualization reagents and/or terminators.

In one embodiment, the antibody in the kit of the invention is detectably labeled. Any label and labeling method known to those skilled in the art may be used. Commonly used labels may include enzymes (e.g., horseradish peroxidase, beta-galactosidase, alkaline phosphatase, etc.), radioisotopes (e.g., horseradish peroxidase, beta-galactosidase, etc.), and the like³²P or¹²⁵I) Etc., biotin, digoxin, colloidal metals (e.g., colloidal gold, etc.), fluorescent dyes (e.g., fluorescein, rhodamine, texas red, etc.), chemiluminescent compounds, or bioluminescent compounds (e.g., dioxetane, luminol, acridinium, etc.). Any labeling step well known in the art may be used, such as covalent coupling of an enzyme or biotin group, iodination, phosphorylation, biotinylation, and the like.

In some embodiments, one or more of the other reactants required for conducting the assay may also be detectably labeled.

The present invention also includes a method of detecting the presence of lipoprotein (a) in a subject, the method comprising: contacting a biological sample from a subject with at least one antibody or antigen-binding fragment thereof of the invention under conditions and for a time sufficient to detect binding of said antibody or antigen-binding fragment thereof to an antigenic determinant, to determine the presence in said biological sample of a molecule naturally present in soluble form in the sample and having an antigenic determinant reactive with said at least one antibody or antigen-binding fragment thereof.

In one embodiment, the antibody or antigen-binding fragment thereof is detectably labeled. In another embodiment, the antibody or antigen-binding fragment thereof is not detectably labeled, and wherein the detection of binding of the antibody or antigen-binding fragment thereof to an antigenic determinant is indirect.

The present invention also includes a method of detecting the presence of lipoprotein (a) in a subject, the method comprising: contacting a biological sample from the subject with at least one antibody or antigen-binding fragment thereof of the invention conjugated to a microsphere under conditions and for a time sufficient for the antibody or antigen-binding fragment thereof to specifically bind to the antigenic peptide and thereby form a turbidity-producing complex, and thereby detecting the presence of lipoprotein (a).

The present invention also includes a method of detecting the presence of lipoprotein (a) in a subject, the method comprising: contacting a biological sample from a subject with at least one immobilized first antibody or antigen-binding fragment thereof specific for an antigenic peptide of the invention to determine the presence of the molecule in the sample, said contacting being conducted under conditions and for a time sufficient for said first antibody or antigen-binding fragment thereof to specifically bind to the antigenic peptide and thereby form an immune complex; removing components of the sample that do not specifically bind to the first antibody; and contacting the immune complex with at least one second antibody or antigen-binding fragment thereof specific for the antigenic peptide of the present invention, wherein the antigen-binding site of the second antibody or antigen-binding fragment thereof does not competitively inhibit the antigen-binding site of the immobilized first antibody or antigen-binding fragment thereof, under conditions and for a time sufficient to detect specific binding of the second antibody or antigen-binding fragment thereof to the antigenic peptide, and thereby detect the presence of lipoprotein (a). In one embodiment, the immobilized first antibody or antigen-binding fragment thereof is selected from an antibody or antigen-binding fragment thereof that specifically binds to an antigenic peptide set forth in SEQ ID NO. 3. In one embodiment, the second antibody or antigen-binding fragment thereof is selected from the group consisting of an antibody or antigen-binding fragment thereof that specifically binds to an antigenic peptide set forth in SEQ ID NO. 1 or 2.

The present invention also discloses a method of detecting lipoprotein (a) in a subject, the method comprising: detecting lipoproteins (a) in a biological sample obtained from a subject; contacting the biological sample with an antibody or antigen-binding fragment thereof of the invention under conditions and for a time sufficient to form an antibody/antigen complex; and displaying the presence or absence of the complex.

In the present invention, the biological sample includes serum and plasma.

Detection methods include, but are not limited to, autoradiography, turbidimetry, fluorescence microscopy, direct and indirect enzymatic reactions, radioisotopes or non-radioisotopes, and the like. These methods include, inter alia, immunoturbidimetry, latex-enhanced transmission turbidimetry, Western blotting, overlay assays, RIA (radioimmunoassay) and IRMA (immunoradioimmunoassay), GIA (colloidal gold immunoassay), EIA (enzyme immunoassay), ELISA (enzyme-linked immunosorbent assay), FIA (fluorescent immunoassay), and CLIA (chemiluminescent immunoassay).

Based on studies on the complete amino acid sequence of apo (a) (UniProtKB/Swiss-Prot: P08519.1), the present inventors found that the CYHGDGQSYRGSFSTT fragment in KIV-6 was able to screen for non-KIV-2 specific lipoprotein (a) antibodies as antigenic peptide, which is also present in KIV-7; furthermore, it was also found that CYRGDGQSYRGTLSTT fragment in KIV-8 can be used as an antigenic peptide to screen for non-KIV-2 specific lipoprotein (a) antibodies; the CYHGDGRSYRGISSTT fragment in KIV-9 can be used as an antigenic peptide to screen for non-KIV-2 specific lipoprotein (a) antibodies.

When used as an antigen, the present invention also provides a fusion protein comprising the antigenic peptide of the present invention. The antigenic peptide of the present invention can be coupled with a protein carrier (also called carrier protein) commonly used in the field of immunology to form a fusion protein so as to more fully induce immune reaction. Exemplary protein carriers may be KLH, BSA or OVA.

The antigenic peptide of the present invention may be present in multiple copies.

The invention also relates to nucleic acids encoding antigenic peptides, e.g. for use in the production of the antigenic peptides or fusion proteins comprising the antigenic peptides of the invention. Nucleic acids encoding antigenic peptides of the present invention include, but are not limited to: the coding sequence of the antigenic peptide itself; a coding sequence for an antigenic peptide and additional coding sequences; the coding sequence (and optional additional coding sequences) and non-coding sequences of the antigenic peptide, for example, may additionally include, but are not necessarily limited to, one or more regulatory nucleic acid sequences, which may be regulated or regulatable promoters, enhancers, other transcriptional regulatory sequences, repressor binding sequences, translational regulatory sequences, or any other regulatory nucleic acid sequence. Thus, a nucleic acid encoding an antigenic peptide encompasses a nucleic acid that includes only the coding sequence for the polypeptide as well as a nucleic acid that includes additional coding and/or non-coding sequences.

Equivalent expression vectors are also encompassed by the present invention. The appropriate nucleic acid sequence may be inserted into any of a variety of well-known expression vectors suitable for the host cell of choice using a variety of methods. Generally, the nucleic acid sequence is inserted into the appropriate restriction endonuclease site using methods known in the art. Standard techniques for cloning, isolation, amplification and purification, enzymatic reactions involving DNA ligases, DNA polymerases, restriction endonucleases and the like, and various isolation techniques, terms known and commonly used by those skilled in the art.

The antigenic peptides of the present invention can be produced using expression vectors and host cells in a variety of expression systems, such as prokaryotic and eukaryotic expression systems. As will be described below with reference to mammalian expression systems, host cells may include the COS-7 cell line of monkey kidney fibroblasts and other cell lines capable of expressing compatible vectors, such as the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors should contain an origin of replication, a suitable promoter and enhancer, and any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from, for example, SV40 splicing and polyadenylation sites can be used to provide the required non-transcribed genetic elements. The constructs may be introduced into the host cell by a variety of methods familiar to those skilled in the art, including, but not limited to, for example, calcium phosphate transfection, DEAE-dextran mediated transfection, or electroporation.

The antigenic peptide of the invention may be an unmodified polypeptide, or may be a post-translationally modified polypeptide, for example by glycosylation, phosphorylation, lipoylation (including glycosylphosphatidylinositol anchor modifications, etc.), phospholipase cleavage (such as phosphatidylinositol-specific phospholipase c mediated hydrolysis, etc.), protease cleavage, dephosphorylation, or any other type of post-translational modification of a protein, for example, a modification involving the formation or cleavage of a covalent chemical bond.

The embodiments of the present invention will be described in detail with reference to examples, which do not indicate specific conditions, and are performed according to conventional conditions or conditions suggested by manufacturers. The reagents or apparatus used are not indicated to the manufacturer, but are conventional products which are commercially available.

EXAMPLE 1 prediction of antigenic peptide sequences

The complete amino acid sequence of apo (a) was obtained by Genebank (UniProtKB/Swiss-Prot: P08519.1), and the antigenic peptides were predicted from the presumed three-dimensional structure of KIV, the specific sequences are shown in Table 1 below.

TABLE 1

EXAMPLE 2 preparation of antigen solution

First, antigenic peptides of KIV-6/7, KIV-8 and KIV-9 regions were synthesized by a polypeptide synthesis method, respectively.

Subsequently, according to the specific procedure of carboxyl and amino coupling in Bioconjugate technologies, Elsevier adaptive press,2 nd edition 2008, P755-763, chapter 19, the protein carrier KLH was activated with EDC, and then the antigenic peptides of KIV-6/7, KIV-8 and KIV-9 regions were coupled to the protein carrier, respectively. The prepared solution can be used as an antigen.

Example 3 preparation of polyclonal antibody specifically recognizing KIV-6/7

Immunization procedure：

1) Taking 3 adult New Zealand rabbits as # A1- # A3;

first immunization: the intradermal multipoint injection comprises two sides of the back, the sole, the periphery of lymph nodes, the back of an ear and the like, the total injection amount of 1 animal is about 8-10 points, and the total injection amount of antigen is 10-100 mu g;

and (3) second immunization: carrying out second immunization at intervals of 10-20 days, and selecting subcutaneous multipoint injection, wherein the total amount of antigen injection is 10-100 ug;

and (3) third immunization: carrying out third immunization at intervals of 10-15 days, and selecting subcutaneous injection, wherein the total amount of antigen injection is 10-100 ug;

the fourth immunization: and performing fourth boosting immunization at intervals of 7-10 days, and selecting subcutaneous injection with the total amount of 10-100 ug of antigen injection.

The sera obtained after immunization of animals using the above method were subjected to titer detection:

1. fixing known antigen on a plastic hole plate, and washing off redundant antigen after completion;

2. adding different dilution gradients of the sample to be tested into each hole, wherein if the sample contains primary antibodies to be tested, the primary antibodies can be specifically bonded with the antigens on the plastic hole plate;

3. washing off redundant samples to be detected, adding a secondary antibody with horseradish peroxidase, and bonding the secondary antibody with the primary antibody to be detected;

4. washing off redundant non-bonded secondary antibody, adding enzyme substrate to make enzyme color, measuring light absorption value (OD450 value) in plastic disc by enzyme-linked immunosorbent assay (ELISA reader) to evaluate the content of colored final product.

KIV-6/7 antigen peptide, KIV-2 antigen peptide and natural lipoprotein (a) were selected as antigens, and serum which was positive to both KIV-6/7 antigen peptide and natural lp (a) and negative to KIV-2 antigen peptide was selected as the target antibody, and the results are shown in Table 2 below.

TABLE 2

Note: the titer is the highest dilution satisfying that the signal value/blank value is more than or equal to 2.1; "blank" is the average of two replicate measurements of OD 450; NC is negative control; in the "envelope" column, A is KIV-6/7 antigen peptide, B is KIV-2 antigen peptide and C is lipoprotein (a).

As shown in Table 2, the polyclonal antibody # A1 was high in potency against KIV-6/7 antigen peptide, high in potency against lipoprotein (a), and low in potency against KIV-2 antigen peptide.

Protein A affinity purification of the polyclonal antibody # A1 was carried out according to the purification procedures described in the handbook of Protein experiments (Mark Page and Robin Thorpe, purification using Protein A or Protein G, Humana Press, second edition 2002, Chapter 42: 993-.

Example 4 preparation of lipoprotein (a) detection kit Using polyclonal antibody # A1

Reagent 1: 0.2M phosphoric acid buffer, pH 7.5, 1% BSA;

reagent 2: the purified polyclonal antibody # a1 finally prepared in example 3 was chemically coupled to the carboxyl microspheres using the carboxyl microsphere two-step coupling process described below:

1. taking 1ml of commercial latex microspheres (with the particle size of 100-200 nm and the concentration of 10%) to 0.05M of 0.5-7.5 MES buffer, and fully and uniformly mixing for 10 min;

2. adding an activating agent EDC with 3 times of carboxyl molar times into the solution within 5s, and activating for 15-30 min;

3. centrifuging 5000-1000 g to remove supernatant, and resuspending the precipitate to 0.05-0.2M potassium phosphate buffer solution with pH of 6.5-8.0;

4. adding the antibody with saturated adsorption capacity into the activated microspheres, and stirring at medium speed for 2-4 h;

5. adding a blocking agent (0.05-0.2M potassium phosphate buffer solution with the pH value of 8.0, 3g/L BSA, 5g/L arginine and 2g/L Tween 80) into the solution after the reaction in the step 4;

and (3) carrying out ultrasonic treatment (power is 50%) on the solution obtained in the step (5), wherein the ultrasonic treatment is carried out for 10min, the absorbance is within 10000, and the obtained solution is used as a reagent 2.

Example 5 methodological alignment of polyclonal antibody # A1 with commercial reagents

To evaluate the effect of the lipoprotein (a) detection kit based on polyclonal antibody #1 prepared in example 4, it was compared with the methodology of the Roche reagent cobas701 system, which had been traced to nmol/L.

The parameters of the Roche reagent on the cobas701 were measured according to the instructions of the Roche lp (a) kit.

The detection process of the kit on the Micheli BS-2000 biochemical analyzer is as follows:

1) mixing 2ul sample with 180ul R1, and incubating at 37 deg.C;

2) adding 45ul of R2, mixing, and incubating at 37 deg.C;

3) the following parameters were used for the measurement

Dominant wavelength: 605nm, sub-wavelength: 800nm, blank time: 18-18, end time: 32-33. The results are shown in FIG. 1.

As shown in FIG. 1, the lipoprotein (a) detection kit based on the polyclonal antibody # A1 and R of the Roche reagent cobas701 system²At 0.9887, the two are highly comparable.

Example 6 evaluation of the Performance of polyclonal antibody # A1

To further examine whether the test kit for detecting lipoprotein (a) based on polyclonal antibody #1 prepared in example 4 can satisfy the performance requirements of the reagents, according to the test method of the test kit of the present invention in example 5, 80 international nmol/L traceable reference substances were used for detection, and the test was repeated 2 times to obtain an average value and calculate the absolute deviation and the relative deviation (target ratio to the reference substance) thereof.

Each point in fig. 2 corresponds to a reference substance, and the absolute deviation is independent of the particle size as can be seen from the distribution of the points. As can be seen from FIG. 3, when the sample concentration is 0 to 50nmol/L, the absolute deviation is within 5 nmol/L; when the sample concentration is 50-100 nmol/L, the absolute deviation is within 10 nmol/L. As can be seen from FIG. 4, when the sample concentration is greater than 100nmol/L, the relative deviation is within 10%. Therefore, the absolute deviation and the relative deviation of the detection kit prepared by using the polyclonal antibody # A1 can meet the requirements of reagent performance.

Example 7 preparation of polyclonal antibody specifically recognizing KIV-8

First, 4 New Zealand rabbits were immunized using the KIV-8 antigen solution prepared in example 2 according to the immunization procedure in example 3. The sera obtained from the immunized animals were then tested according to the titer detection method in example 3, and the results are shown in table 3 below.

TABLE 3

Note: the titer is the highest dilution satisfying that the signal value/blank value is more than or equal to 2.1; "blank" is the average of two replicate measurements of OD 450; NC is negative control; in the "envelope" column, A is KIV-8 antigen peptide, B is KIV-2 antigen peptide and C is lipoprotein (a).

As shown in Table 3, polyclonal antibodies # B2 and # B4 had high titers for the KIV-8 antigen peptide, high titers for lipoprotein (a), and low affinity for the KIV-2 antigen peptide.

Similarly, the polyclonal antibody # B4 was subjected to Protein A affinity purification according to the manual of Protein experiments.

Example 8 preparation of lipoprotein (a) detection kit Using polyclonal antibody # B4

Reagents 1 and 2 for lipoprotein (a) detection were prepared using polyclonal antibody # B4 as described in example 4.

Example 9 methodological alignment of polyclonal antibody # B4 with commercial reagents

In order to evaluate the effect of the lipoprotein (a) detection kit based on polyclonal antibody #1 prepared in example 4, it was compared with the methodology of the Roche reagent cobas701 system, which had been traced to nmol/L, according to the method described in example 5, and the results are shown in FIG. 5.

As shown in FIG. 5, the R between the lipoprotein (a) detection kit based on the polyclonal antibody # B4 and the Roche reagent cobas701 system²At 0.9924, the two are extremely comparable.

Example 10 evaluation of the Performance of polyclonal antibody # B4

To further examine whether the test kit for lipoprotein (a) based on polyclonal antibody # B4 prepared in example 8 can satisfy the performance requirements of the reagents, the test was performed according to the method of example 6, and the results are shown in FIGS. 6 to 8.

Each point in fig. 6 corresponds to a reference substance, and as can be seen from the distribution of the points, the absolute deviation is very independent of the particle size. As can be seen from FIG. 7, when the sample concentration is 0 to 50nmol/L, the absolute deviation is within 5 nmol/L; when the sample concentration is 50-100 nmol/L, the absolute deviation is basically within 10 nmol/L. As can be seen from FIG. 8, when the sample concentration is greater than 100nmol/L, the relative deviation is substantially within 10%. Therefore, the absolute deviation and the relative deviation of the detection kit prepared by using the polyclonal antibody # B4 can meet the requirement of tracing the performance of the reagent.

EXAMPLE 11 preparation of polyclonal antibody that specifically recognizes KIV-9

First, 6 New Zealand rabbits were immunized using the KIV-9 antigen solution prepared in example 2 according to the immunization procedure in example 3. The sera obtained from the immunized animals were then tested according to the titer detection method in example 3, and the results are shown in table 4 below.

TABLE 4

Note: the titer is the highest dilution satisfying that the signal value/blank value is more than or equal to 2.1; "blank" is the average of two replicate measurements of OD 450; in the "envelope" column, A is KIV-9 antigen peptide, B is KIV-2 antigen peptide and C is lipoprotein (a).

As shown in Table 4, the polyclonal antibody # C3 was high in potency against KIV-9 antigen peptide, high in potency against lipoprotein (a), and low in potency against KIV-2 antigen peptide.

Similarly, Protein a affinity purification was performed on the polyclonal antibody # C3 described above according to the manual of Protein experiments.

EXAMPLE 12 preparation of lipoprotein (a) detection kit Using polyclonal antibody # C3

Reagents 1 and 2 for lipoprotein (a) detection were prepared using the polyclonal antibody # C3 as described in example 4.

Example 13 methodological alignment of polyclonal antibody # C3 with commercial reagents

To evaluate the effect of the lipoprotein (a) detection kit based on polyclonal antibody # C3 prepared in example 12, it was aligned with the roche reagent cobas701 system, which was traced to nmol/L, according to the method in example 5, and the results are shown in fig. 9.

As can be seen from FIG. 9, the lipoprotein (a) detection kit based on the polyclonal antibody # C3 and R of the Roche reagent cobas701 system²At 0.991, the two are extremely comparable.

Example 14 evaluation of the Performance of polyclonal antibody # C3

To further examine whether the test kit for lipoprotein (a) based on polyclonal antibody # C3 prepared in example 12 can satisfy the performance requirements of the reagents, the test was performed according to the method of example 6, and the results are shown in fig. 10 to 12.

Each point in fig. 10 corresponds to a reference material, and the absolute deviation is independent of the particle size, as can be seen from the distribution of the points. As can be seen from FIG. 11, when the sample concentration is 0 to 50nmol/L, the absolute deviation is within 5 nmol/L; when the sample concentration is 50-100 nmol/L, the absolute deviation is within 10 nmol/L. As can be seen from FIG. 12, when the sample concentration is greater than 100nmol/L, the relative deviation is within 10%. Therefore, the absolute deviation and the relative deviation of the detection kit prepared by using the polyclonal antibody # C3 can meet the requirements of reagent performance.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and all equivalent modifications made by the present invention in the light of the present specification, or directly/indirectly applied to other related technical fields are included in the scope of the present invention.

Sequence listing

<110> Shenzhen Merrill biomedical electronics Limited

Antigenic peptide of <120> lipoprotein (a), antibody thereof and use thereof

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<170> PatentIn version 3.5

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<213> Artificial Synthesis

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Cys Tyr Arg Gly Asp Gly Gln Ser Tyr Arg Gly Thr Leu Ser Thr Thr

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Claims

1. An antibody or antigen binding fragment thereof that specifically binds to one or more KIV antigen peptides selected from the group consisting of those shown in SEQ ID NOs 1-3.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the KIV antigen peptide shown as SEQ ID NO. 3 is specifically bound.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a bispecific antibody, a chimeric antibody, a humanized antibody, and a single chain antibody.

4. The antibody or antigen-binding fragment thereof of claim 3, wherein the antibody is a monoclonal antibody.

5. The antibody or antigen-binding fragment thereof of claim 3, wherein the antibody is a polyclonal antibody.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody is of murine, rabbit or ovine origin.

7. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 6.

8. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 6.

9. The kit of claim 8, comprising:

1) an antibody or antigen-binding fragment thereof as defined in any one of claims 1 to 6, and which is coated on a solid support;

2) an antibody or antigen-binding fragment thereof as defined in any one of claims 1 to 6 and which carries a detectable label;

3) an antibody or antigen-binding fragment thereof, as defined in any one of claims 1 to 6, which is not coated on a solid support and which does not carry a detectable label; or

4) A first antibody or antigen-binding fragment thereof, as defined in any one of claims 1 to 6; and a second antibody or antigen-binding fragment thereof, as defined in any one of claims 1 to 6, and wherein the first antibody or antigen-binding fragment thereof binds a KIV antigen peptide that is different from the KIV antigen peptide of the second antibody or antigen-binding fragment thereof.

10. Use of an antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 6 in the manufacture of a kit for use in a method for assessing cardiovascular disease, the method comprising the steps of:

11. The use of claim 10, wherein the antibody or antigen-binding fragment thereof is coated on a solid support, the antibody or antigen-binding fragment thereof carries a detectable label, or the antibody or antigen-binding fragment thereof is not coated on a solid support and does not carry a detectable label.

12. An antigenic peptide, the amino acid sequence of which is shown in one or more of SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3.

13. The antigenic peptide of claim 12, having the amino acid sequence set forth in SEQ ID NO 3.

14. A fusion protein comprising the antigenic peptide of claim 12 or 13.

15. The fusion protein of claim 14, wherein the fusion protein further comprises a polypeptide selected from an affinity tag polypeptide, a protein carrier, or a polypeptide that targets the fusion protein to a predetermined location.

16. A nucleic acid molecule encoding the antigenic peptide of claim 12 or 13 or the fusion protein of claim 14 or 15.

17. An expression vector comprising the nucleic acid molecule of claim 16.

18. A host cell comprising the nucleic acid molecule of claim 16 or the expression vector of claim 17.

19. A method for obtaining an anti-lipoprotein (a) antibody that is not specific for KIV-2, using as an antigen the antigenic peptide of claim 12 or 13 or a fusion protein of the antigenic peptide of claim 14 or 15 and a protein carrier.